The ermE gene was cloned from Streptomyces erythraeus into Escherichia coli on a series of plasmids. When transcribed from the lac promoter, ermE conferred high-level resistance to erythromycin and other macrolide-lincosamide-streptogramin-B (MLS) antibiotics. A methylase activity capable of N6-mono- and N6,N6-dimethylation of adenine residues in E. coli rRNA was detected in extracts of MLS-resistant cells. In addition, rRNA extracted from MLS-resistant E. coli contained N6-mono- and N6,N6-dimethylated adenine residues.